Pol6 polymerase variants

ABSTRACT

The present disclosure provides variant Pol6 polymerase polypeptides, compositions comprising the Pol6 variant polypeptides, and methods for using the variant Pol6 polypeptides for determining the sequencing of nucleic acids, for example, by nanopore sequencing. The variant Pol6 polymerases possess decreased rates of dissociation of template from the polymerase-template complex, which result in increased processivity relative to the parental Pol6 polypeptides from which they are derived.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of PCT/EP2017/073767, filed Sep. 20, 2017, and claims the benefit of United States Provisional Patent Application No. U.S. 62/398,407, filed Sep. 22, 2016, United States Provisional Patent Application No. U.S. 62/414,669, filed Oct. 29, 2016, and United States Provisional Patent Application No. U.S. 62/440,103, filed Dec. 29, 2016, the content of each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

Provided herein, among other things, are modified DNA polymerases containing amino acid alterations based on mutations identified in directed evolution experiments designed to select enzymes that are better suited for applications in recombinant DNA technologies.

BACKGROUND

DNA polymerases are a family of enzymes that use single-stranded DNA as a template to synthesize the complementary DNA strand. In particular, DNA polymerases can add free nucleotides to the 3′ end of a newly-forming strand resulting in elongation of the new strand in a 5′ to 3′ direction. Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For example, many DNA polymerases have 3′→5′ exonuclease activity. These polymerases can recognize an incorrectly incorporated nucleotide and the 3′→5′ exonuclease activity of the enzyme allows the incorrect nucleotide to be excised (this activity is known as proofreading). Following nucleotide excision, the polymerase can re-insert the correct nucleotide and replication can continue. Many DNA polymerases also have 5′→3′ exonuclease activity.

Polymerases have found use in recombinant DNA applications, including nanopore sequencing. However, a DNA strand moves rapidly at the rate of 1 μs to 5 μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. Therefore, the use of detectable tags on nucleotides may be used in the sequencing of a DNA strand or fragment thereof. Thus, there is a not only a need to control the rate of DNA being sequenced but also provide polymerases that have improved properties (relative to the wild-type enzyme) such as incorporation of modified nucleotides, e.g., polyphosphate nucleotides with or without tags.

SEQUENCE LISTING INCORPORATION BY REFERENCE

A Sequence Listing filed herewith, entitled “33884US4_Sequence_Listing_ST25,” created on Mar. 8, 2019, with a file size of 17,885 bytes, is hereby incorporated by reference.

BRIEF SUMMARY

The present invention provides modified DNA polymerases (e.g., mutants) based on directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications, e.g., catalyzing incorporation of modified polyphosphate nucleotides, e.g., tagged nucleotides, under high salt concentrations.

The present invention is directed to, among other aspects, modified DNA polymerases having improved extension rate and dissociation rate for polyphosphate nucleotides with or without tags in high salt conditions.

One aspect of the present invention provides advanced polymerase mutations selected for their extension rate and nucleotide dissociation rate for polyphosphate nucleotides through several generation of mutagenesis on wild-type (WT) Pol6 (SEQ ID NO: 1; wild-type pol6 without His-Tag).

The inventors herein discovered that several mutants were found to have improved extension rate as shown in Fluorescence Resonance Energy Transfer (FRET)-based displacement assays using polyphosphate nucleotides with or without tags. See FIG. 1.

In some embodiments, the modified polymerase has a k_(cat) that is greater than the parental polymerase. In some embodiments, the modified polymerase has a k_(off) that is less than the parental polymerase. In some embodiments, the modified polymerase has a k_(cat)/k_(off) (i.e., a ratio) that is at least 1.5, 2.0 or 2.5 times greater than the parental polymerase.

In an embodiment of the invention there is a variant DNA polymerase comprising at least one alteration at a position corresponding to one or more of Y30, I42, D44, E46, C48, G62, G64, W127, G146, T162, V164, I172, I178, S198, L206, F209, A218, Q221, F222, N224, Y225, V226, H227, F233, Y234, K235, M236, G237, D241, Y242, D243, D263, I264, Y265, M266, R268, E269, V270, G278, E283, L284, P285, M289, R290, A292, S293, S294, 1295, A296, F297, N298, V299, E311, E312, Y314, Y338, V350, C359, S360, L361, S366, S369, M371, A372, Y373, K374, F376, K380, V382, R388, P390, E396, Y398, L399, I400, E401, F404, F406, K410, H411, Y414, A415, L416, D417, I418, S430, P431, I432, G438, D448, K456, L458, K462, T464, N468, V469, V470, L471, V474, E475, Y476, E477, F478, W479, K481, H482, F483, E502, G505, L506, I508, S510, Y514, K518, F521, K522, P523, Y524, V525, D526, H527, F528, T529, K530, M531, V533, E534, N535, K537, L538, G539, K541, P542, L543, T544, N545, Q546, A547, L549, L551, N552, G553, A554, Y555, G556, K557, F558, T560, K561, Q562, E566, E585, E587, G588, E590, P594, G603, W608, A610, V615, L621, C623, D624, I628, Y629, C630, N631, R632, V634, N635, I638, E639, D640, M641, N642, A643, I644, T647, K650, T651, I652, L653, K655, D657, V658, E659, K672, E680, K682, T683, D684, L685, K686, C687, C688, S692, D693, A694, R695, K696, I697, I698, L708, V712, R718, I722, G724, F732, M738, and combinations thereof of SEQ ID NO:2 (Pol6 (with His tag)).

In an embodiment, the variant polymerase having altered enzyme activity as compared to SEQ ID NO: 1 or 2, or a parental polymerase is selected from Y30A/C/G/V, I42E/L, D44T, E46W, C48G, G62N/E/T, G64A, W127L/S/R/V/G/C/P/Q/A/E, G146K/L, T162C/R/V, V164Q/R/T, I172A, I178A, S198F/L/Q/W/Y/V, L206S, F209I/R/T/V, A218R, Q221F/M/R/W/G/L/E/Y/K, F222U/M/Q/T, H223L, N224K/R/T, Y225T, V226T, H227E/G/A/Q/T, F233E/T/W/P/S, Y234R, K235S/Q/N/F/W/P, M236K, G237A, D241W/H, D243P, D263Q/W/L, I264Y, Y265E, M266V/W, R268E, E269I, V270A/S, G278V/E/M/R/U/P, E283G, L284R/Y, P285U/C/Q/R/S/T, M289P, R290V/C, A292S/U/T, S293G/Q/R/T/M/W/A, S294Y/T/R/M/L/K/G/F/E, I295A/L, A296M, F297M/T/L, N298K/U/R/W/G, V299Y/R/E/A/F/K, E311N/K/H, E312R/U/K, Y314W, Y338L, V350R/L/M, C359T, S360A/F, L361A/M/F, Y367F, S369G/F/Y/K/A, Q370A, M371L, A372G/L, Y373T/R/A, K374H/P/N/F/G/W, F376L/V, K380R, V382L/I, R388P/S, P3901/V, E396N/F, Y398A/P, L399G, I400P/M/L, E401I, F404L, F406K/Y, K410V, H411P, Y414P/R/I/Q/S/T/K/G, A415P, L416W, D417E, I418Q/H, S430P, P431G, I432F, G438R/U/P/Q/C, D448L, K456R, L458P/R, K462G, T464P, N468G, V469E/R/S/T/Y, V470L/I/M/Q/H/A/F, L471C/E/H/K/Q/R/S/V/Y, V474G, E475D/F/G/H/L/N/Q/S/T/V, Y476L, E477G, F478Y/H, W479K, K481L, H482T/G/D/R/W, F483G/N/Q/S/T, E502P/A, G505C/R, L506T/G/C, I508M/P, S510P/F/L, Y514W, K518W, F521M/L, K522R/Q, P523Y/T/K/G/E/R/M/L/Q/A, Y524A/F/W, V525M, D526Q/E, H527F/M/L/R, F528Q/L/Y, K530T/G/F/E/W/Y/R/Q, M531R/L/A, V533A, E534R, N535Y, K537L/E, L538R/Q/K/A, K541R/G/A/Q/W, L543A/K/T/R, T544A, N545Y, Q546A, L549T/R/A, L551M, G552T, G553T/Q/K/A/Y/R/M, A554Q′T, Y555A, G556R, K557W/R/E/Q, F558M, T560Y/R/M/G/F/K/A/L, K561A/Q/M/G/Y, Q562K/R/G, E566Q/Y/G/F, E587K/T, G588E/V/Q, E590R/W, P594A/V, G603Y/L/A, W608R, A610T/C/S/V/V/M, L621W, C623A/G/T, D624L, I628M, Y629K/W, C630A/Q, N631L/T/W/A, R632K, V634L, I638A/E/G/L/M/Q/Y/F/K, E639F/T/W/Y, D640A/E/K/QT, M641L, N642E/F/K/Q/T/W/Y/M, A643K/R/T/Y/L, I644A, T647A/E/F/G/L/M/Q/W/Y/K, K650Q/R/Y/A, T651M, I652A/E/G/K/M/Q/R/W/L, L653M/A, K655A/F/G/M/W/Y, D657E/R, V658F/M/Y/T, E659K/T, K672R/T, E680A/Q/K/F/Y/W/M, K682F/M/Y/V/I, T683F/W, D684F/K/M/Y/G/W/E/T/Q, L685A, K686R/M/T, C687A, C688A/S, S692F/K/R/Y/D/L, D693P/R/M/A/K/Y/W, A694L/M/T/Y/F, R695K, K696Q, I697A, I698L, L708H/P/Y, V712C, R718K, I722R, G724Y, F732Y, M738W/V, and combinations thereof.

In one embodiment there is provided a modified DNA polymerase having a DNA polymerase activity comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 90% or at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 1 or 2.

In some embodiments, the parental polymerase is wild-type Pol6 (SEQ ID NO:1). In some embodiments, the parental polymerase is Pol6 comprising a His-tag (SEQ ID NO:2). In various embodiments, the parental polymerase is a pol6 selected from

-   -   1) A547F;     -   2) A547F+L206S;     -   3) A547F+F209I;     -   4) A547F+E283G;     -   5) A547F+P285L;     -   6) A547F+E311K;     -   7) A547F+L361F;     -   8) A547F+L471K;     -   9) A547F+V474G;     -   10) A547F+H482G;     -   11) A547F+T162C/R/V;     -   12) A547F+V164Q/R/T;     -   13) A547F+E311H;     -   14) A547F+E312R/L/K;     -   15) A547F+Y314W;     -   16) A547F+V350R/L/M;     -   17) A547F+Q562K/R/G;     -   18) A547F+E587K/T;     -   19) A547F+P594A/V;     -   20) A547F+W608R;     -   21) A547F+A610T/C/S/V/M;     -   22) N535L+N545K+T651 Y;     -   23) S366A+N535L+I652Q;     -   24) S366A+T529M+N535L;     -   25) pol6 S366A+N535L+N545K;     -   26) S366A+N535L+A547M;     -   27) S366A+P542E+I652Q;     -   28) S366A+P542E+N545K;     -   29) S366A+P542E+T651Y;     -   30) P542E+N545K+T651 Y;     -   31) P542E+Q546 W+T651 Y;     -   32) N535L+T651 Y;     -   33) S366A+N535L;     -   34) N535L+N545K+T651Y+T529M;     -   35) N535L+N545K+T651Y+N635D;     -   36) N535L+N545K+T651Y+I652Q;     -   37) S366A+N535L+I652Q+T529M;     -   38) N535L+N545K+T651Y+T647G;     -   39) S366A+N535L+I652Q+A547Y;     -   40) S366A+N535L+A547M+T647G;     -   41) S366A+N535I+I652Q;     -   42) N535I+N545K+T651Y+T529M;     -   43) N535I+N545K+T651Y+N635D;     -   44) N535I+N545K+T651Y+I652Q;     -   45) N535L+N545K+T651Y+T647G+C623G;     -   46) N535L+N545K+T651Y+T647G+I628Y;     -   47) S366A+N535L+A547M+T647G+S360G;     -   48) N535I+N545K+T651Y+I652Q+Y225I;     -   49) N535L+N545K+T651Y+T647G+K655G;     -   50) N535L+N545K+T651Y+T647G+L549Q;     -   51) S366A+N535L+I652Q+A547Y+K655G;     -   52) T647G+A547F+Y225T;     -   53) A547F+A610T+S366A;     -   54) A547F+A610T+Y225I;     -   55) S366A+T647G+A547F;     -   56) T651Y+S366A+A547F;     -   57) T529M+S366A+A547F;     -   58) T647E+S366A+A547F;     -   59) T529M+T647G+A547F;     -   60) N545K+S366A+A547F;     -   61) T647G+A547F+T529M;     -   62) N545K+T647G+A547F;     -   63) T529M+A610T+A547F;     -   64) M641Y+T529M+A547F;     -   65) T647G+C623G+A547F;     -   66) A610T+I295 W+T651 Y;     -   67) V615A+M531Y+T647G;     -   68) T529M+S366A+A547F+N545K;     -   69) T529M+S366A+A547F+N545R;     -   70) T529M+S366A+A547F+N552L;     -   71) T529M+S366A+A547F+Y629W;     -   72) T529M+S366A+A547F+N545L+Y629W;     -   73) T529M+S366A+A547F+N545L+Y225L;     -   74) T529M+S366A+A547F+N545L+Y225F;     -   75) T529M+S366A+A547F+N545L+K655F;     -   76) T529M+S366A+A547F+N545L+N552L;     -   77) T529M+S366A+A547F+N545R+M531A;     -   78) T529M+S366A+A547F+N545R+G539Y;     -   79) T529M+S366A+A547F+N545R+V658L;     -   80) T529M+S366A+A547F+N545L+Y225L+D657R;     -   81) T529M+S366A+A547F+N545L+Y225L+N552L;     -   82) T529M+S366A+A547F+N545L+Y225L+1652G;     -   83) T529M+S366A+A547F+N545L+Y225L+I652Q;     -   84) T529M+S366A+A547F+N545L+Y225L+N552M;     -   85) T529M+S366A+A547F+N545L+Y225L+N552L+Y524F;     -   86) T529M+S366A+A547F+N545L+Y225L+N552L+S369Y;     -   87) T529M+S366A+A547F+N545L+Y225L+N552L+Y524W;     -   88) T529M+S366A+A547F+N545L+Y225L+N552L+K651A;     -   89) T529M+S366A+A547F+N545L+Y225L+N552L+T560M;     -   90) T529M+S366A+A547F+N545L+Y225L+N552L+S293T;     -   91) T529M+S366A+A547F+N545L+Y225L+D657R+T560Y;     -   92) T529M+S366A+A547F+N545L+Y225L+D657R+T560R;     -   93) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G;     -   94) T529M+S366A+A547F+N545L+Y225L+D657R+K561 Y;     -   95) T529M+S366A+A547F+N545L+Y225L+D657R+N224R;     -   96) T529M+S366A+A547F+N545L+Y225L+D657R+I628M;     -   97) T529M+S366A+A547F+N545L+Y225L+D657R+K655A;     -   98) T529M+S366A+A547F+N545L+Y225L+D657R+S2930;     -   99) T529M+S366A+A547F+N545L+Y225L+D657R+Y629W;     -   100) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+I628M;     -   101) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R;     -   102) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+I638E;     -   103) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K655M;     -   104) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K655Y;     -   105) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+V634L;     -   106) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+N224K;     -   107) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+S293Q;     -   108) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+N298K;     -   109) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+S294T;     -   110) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+F521L;     -   111) T529M+S366A+A547F+N545L+Y225L+D657R+S692F;     -   112) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+K682Y;     -   113) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+D684G;     -   114) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+T544A;     -   115) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+I 628M;     -   116) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+N642E;     -   117) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+S692F;     -   118) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+C630A;     -   119) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+I 638G;     -   120) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S294L;     -   121) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S294T;     -   122) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+I628M;     -   123) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+K655A;     -   124) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+Q221R;     -   125) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+G553A;     -   126) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+T647F;     -   127) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+K682Y;     -   128) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y;     -   129) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692R;     -   130) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+S293G;     -   131) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+S293Q;     -   132) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+K655G;     -   133) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+D684Y;     -   134) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+D684M;     -   135) T529M+S366A+A547F+N545L+Y225L+D657R+S692F+T560M;     -   136) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+S293Q;     -   137) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+L361M;     -   138) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+Y629W;     -   139) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+T647Q;     -   140) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+I652Q;     -   141) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+L653M;     -   142) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+L549R;     -   143) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+G553K;     -   144) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+G552T;     -   145) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+1652G;     -   146) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693R;     -   147) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P;     -   148) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+C359T;     -   149) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+P523L;     -   150) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+H527F;     -   151) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+H527R;     -   152) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+K530E;     -   153) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+G603A;     -   154) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+N642M;     -   155) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P+K655A     -   156) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+1652Q;     -   157) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+D693P; and     -   158) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+1652Q+D684M.

In some embodiments, the parental polymerase of 1-158, above, may further comprise a mutation selected from Y242A, E585K, and combinations thereof.

In an embodiment, the variant polymerase is selected from

-   -   1) A547F further comprising one or more mutations selected from         -   a. Y30A/C/G/V;         -   b. I42E/L;         -   c. D44T;         -   d. E46W;         -   e. C48G;         -   f. G62A/E/T;         -   g. G64A;         -   h. W127L/S/R/N/G/C/P/Q/A/E;         -   i. G146K/L;         -   j. T162C/R/V;         -   k. V164Q/R/T;         -   l. I172A;         -   m. I178A;         -   n. S198V;         -   o. F209I/R/T/V;         -   p. A218 R;         -   q. Q221W/R/G/L/E/F;         -   r. F233E/T/W/P/S;         -   s. Y234R;         -   t. K235S/Q/N/F/W/P;         -   u. M236K;         -   v. G237A;         -   w. D241W/H;         -   x. D243P;         -   y. D263Q/W/L;         -   z. I264Y;         -   aa. Y265E;         -   bb. M266V/W;         -   cc. R268E;         -   dd. E269I;         -   e. V270A/S;         -   ff. G278V/E/M/R/L/P;         -   gg. L284R/Y;         -   hh. P285U/C/Q/R/S/T;         -   ii. M289P;         -   jj. R290V/C;         -   kk. A292S/L/T;         -   ll. E311N/H;         -   mm. E312R/L/K;         -   nn. Y314W;         -   oo. V350R/L/M;         -   pp. K374H/P/N/F/G/W;         -   qq. F376L/V;         -   rr. K380R;         -   ss. V382L/I;         -   tt. R388P/S;         -   uu. P390L/V;         -   vv. E396N;         -   ww. Y398A/P;         -   xx. L399G;         -   yy. I400P/M/L;         -   zz. E401I;         -   aa. F404L;         -   bbb. K410V;         -   ccc. H411P;         -   ddd. Y414P/R/I/Q/S/T/K/G;         -   eee. A15P;         -   fff. L416W;         -   ggg. D417E;         -   hhh. I418Q/H;         -   iii. S430P;         -   jjj. P431 G;         -   kkk. I432F;         -   lll. G438R/L/P/Q/C;         -   mmm. K456R;         -   nnn. L458P/R;         -   ooo. K462G;         -   ppp. T464P;         -   qqq. N468G;         -   rrr. V469E/R/S/T/Y;         -   sss. V470L/I/M/Q/H/A/F;         -   ttt. L471C/E/H/K/Q/R/S/V/Y;         -   uuu. E475D/F/G/H/L/N/Q/S/T/V;         -   vvv. Y476L;         -   www. E477G;         -   xxx. F478Y/H;         -   yyy. W479K;         -   zz. K481L;         -   aa. H482T/D/R/W;         -   bbbb. F483G/N/Q/S/T;         -   cccc. E502P/A;         -   dddd. G505C/R;         -   eeee. L506T/G/C;         -   ffff. I508M/P;         -   gggg. S510P/F/L;         -   hhhh. Y514W;         -   iiii. K518W;         -   jjjj. N545Y;         -   kkkk. Q562K/R/G;         -   llll. E587K/T;         -   mmmm. G588E/V/Q;         -   nnnn. E590R/W;         -   oooo. P594A/V;         -   pppp. W608R;         -   qqqq. A610T/C/S/V/M;         -   rrrr. K672R;         -   ssss. K682F/M/Y/V/I;         -   tttt. D684G;         -   uuuu. L685A;         -   vvvv. C688A/S;         -   wwww. S692F/D/R/L;         -   xxxx. D693P/R;         -   yyyy. K696Q;         -   zzzz. I698L;         -   aaaaa. L708H/P/Y;         -   bbbbb. V712C;         -   ccccc. R718K;         -   ddddd. I722R;         -   eeeee. G724Y;         -   fffff. F732Y;         -   ggggg. M738W/V     -   2) A547F+L206S, further comprising         -   a. P285L     -   3) A547F+F209I, further comprising         -   a. D448L     -   4) A547F+E283G, further comprising         -   a. L471K     -   5) A547F+P285L, further comprising         -   a. L206S     -   6) A547F+E311K, further comprising         -   a. V474G     -   7) A547F+L361F, further comprising         -   a. H482G     -   8) A547F+L471K, further comprising         -   a. E283G     -   9) A547F+V474G, further comprising         -   a. E311K     -   10) A547F+H482G, further comprising         -   a. L361F     -   11) N545K+T647G+A547F, further comprising one or more mutations         selected from         -   a. Q221W;         -   b. Y225T;         -   c. Y242A;         -   d. F528Q/L;         -   e. K530W;         -   f. T560K/Y;         -   g. I638A;         -   h. L653A;         -   i. D657E/R     -   12) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G, further         comprising one or more mutations selected from         -   a. Y242A;         -   b. S294Y/K;         -   c. I295L;         -   d. N298L;         -   e. C359T;         -   f. L361M/A;         -   g. F521L;         -   h. P523K;         -   i. K530G;         -   j. L538R;         -   k. K541R;         -   l. K557R;         -   m. F558M;         -   n. T560F/R;         -   o. G603L;         -   p. C623A;         -   q. D624L;         -   r. I628M;         -   s. V634L;         -   t. I638E;         -   u. M641L;         -   v. T647G/A/E;         -   w. K655M/A/Y     -   13) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R, further         comprising one or more mutations selected from         -   a. Q221M;         -   b. N224K;         -   c. Y242A;         -   d. S293Q;         -   e. S294T;         -   f. N298K;         -   g. L361M;         -   h. Y373A;         -   i. F521M/L;         -   j. K522R;         -   k. P523E;         -   l. M531L;         -   m. L549A;         -   n. T560R;         -   o. I628M;         -   p. C630A;         -   q. R632K;         -   r. I638G/A/Q;         -   s. N642E;         -   t. T647E;         -   u. K650Q;         -   v. I652G;         -   w. K655F;         -   x. V658F     -   14) T529M+S366A+A547F+N545L+Y225L+D657R+S692F, further         comprising one or more mutations selected from         -   a. S198F/L/Q/W/Y;         -   b. Q221F;         -   c. F222L/T/Q;         -   d. H227E;         -   e. Y242A;         -   f. S293G/Q/R/T;         -   g. F297M;         -   h. S294G/Y;         -   i. V299Y/R/E/A;         -   j. L361M;         -   k. S369G;         -   l. Y373A/R;         -   m. F406K;         -   n. F521M/L;         -   o. K522Q;         -   p. P523G/Y;         -   q. D526Q;         -   r. K530T;         -   s. M531R/L/A;         -   t. L538Q;         -   u. K541R/G;         -   v. T544A;         -   w. Q546A;         -   x. K557W/R;         -   y. F558M;         -   z. T560G/F/Y/M;         -   aa. E566Q/Y/G/F;         -   bb. G603Y;         -   cc. I628M;         -   dd. Y629W;         -   ee. I638G/A/L/E/Y/M;         -   ff. E639F/T/W/Y;         -   gg. D640A/E/K;         -   hh. N642E/Q/T;         -   ii. A643Y/T;         -   jj. I644A;         -   kk. T647Y/M;         -   ll. 1652K;         -   mm. K655G;         -   nn. V658F/M;         -   oo. D684F/K/M/Y     -   15) T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+T544A,         further comprising one or more mutations selected from         -   a. Q221M/R;         -   b. F222M;         -   c. N224R;         -   d. Y242A;         -   e. S293T;         -   f. S294M/L/T/F/Y;         -   g. N298K/L/R;         -   h. P523T;         -   i. G553A/Q;         -   j. K557R;         -   k. E566G;         -   l. I628M;         -   m. Y629K;         -   n. C630Q;         -   o. V634L;         -   p. I638G;         -   q. A643R/K;         -   r. T647W/L/F;         -   s. K655A;         -   t. K682Y;         -   u. S692F/K/R/Y     -   16) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y, further comprising one or more mutations         selected from         -   a. N224T;         -   b. Y242A;         -   c. S293Q;         -   d. S294R/T;         -   e. I295A;         -   f. V299A/R;         -   g. L361M;         -   h. S369F;         -   i. Y373A/T;         -   j. F528Y;         -   k. K530G/E/F;         -   l. L549T/R;         -   m. G553A/K/T;         -   n. A554Q;         -   o. G556R;         -   p. K557R;         -   q. F558M;         -   r. T560F/Y;         -   s. E585K;         -   t. Y629W;         -   u. C630A;         -   v. I638A/E;         -   w. N642E/K/F/Y/W;         -   x. T647G/L/Q/M;         -   y. K650Q;         -   z. T651M;         -   aa. 1652G/A/E/Q/M;         -   bb. L653M;         -   cc. K655G/W;         -   dd. V658Y;         -   ee. D693R/M/P;         -   ff. A694L     -   17) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+I652Q, and further comprising one or more         mutations selected from         -   a. Q221L;         -   b. F222L;         -   c. Y242A;         -   d. Y338L;         -   e. L361F;         -   f. Q370A;         -   g. K537E;         -   h. L543K/R;         -   i. G553A/K/Y/R/T/Q;         -   j. A554T;         -   k. E585K;         -   l. I628M;         -   m. Y629K;         -   n. N631T;         -   o. R632K;         -   p. N642Q/F/Y;         -   q. A643L;         -   r. D684G/F/W/M/Q;         -   s. D693A/K/M/Y     -   18)         T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P,         and further comprising one or more mutations selected from         -   a. N224K;         -   b. H227G/A/Q/T;         -   c. Y242A;         -   d. S293A/Q/T;         -   e. V299F;         -   f. C359T;         -   g. L361M;         -   h. F406Y;         -   i. F521M;         -   j. P523L/E/Q/K/R/T/M/A;         -   k. H527F/LM/R;         -   l. K530G/E;         -   m. N535Y;         -   n. G553A;         -   o. Y555A;         -   p. T560Y;         -   q. E585K;         -   r. G603A;         -   s. C623G;         -   t. Y629K;         -   u. N642M;         -   v. T647K/F/Y;         -   w. 1652G/L/Q/K/R/W/M;         -   x. K655A;         -   y. D684F/Y/W/M;         -   z. K686R     -   19)         T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P+V299F,         and further comprising one or more mutations selected from         -   a. Q221K/M;         -   b. H223L;         -   c. S294E/T;         -   d. F297L/M;         -   e. S293G;         -   f. N298G;         -   g. Y338L;         -   h. C359T;         -   i. S360A;         -   j. L361F;         -   k. Y367F;         -   l. S369A;         -   m. E396F;         -   n. K522Q;         -   o. D526E;         -   p. K530R;         -   q. M531A;         -   r. L538A;         -   s. L543T;         -   t. G553K/R/M;         -   u. K557R/E/Q;         -   v. T560L;         -   w. L621W;         -   x. C623T;         -   y. C630Q;         -   z. N631A;         -   aa. I638K/F/E;         -   bb. D640E/Q/T;         -   cc. V658T/M;         -   dd. E680A/Q/K/F/Y/W/M;         -   ee. T683F/W;         -   if. D684E/T/K;         -   gg. L685A;         -   hh. K686M;         -   ii. C687A;         -   jj. A694M/T/Y;         -   kk. R695K;         -   ll. I697A     -   20) T529M+S366A+A547F+N545L+Y225L+D657R+K561         G+K541R+T544A+S692Y+I652Q+D684M, and further comprising one or         more mutations selected from         -   a. Q221R;         -   b. N224K;         -   c. Y242K;         -   d. S293Q/G;         -   e. N298L/K/W;         -   f. V299R;         -   g. S360A/F;         -   h. L361F/M/A;         -   i. K522R;         -   j. H527M;         -   k. K530F;         -   l. K537E;         -   m. L543K;         -   n. G553Y/R/T/M;         -   o. A554Q/T;         -   p. E585K;         -   q. G603A;         -   r. Y629W/K;         -   s. N631W;         -   t. I638F;         -   u. N642M;         -   v. K650A;         -   w. K655F;         -   x. K672T;         -   y. K686T;         -   z. D693R/W;         -   aa. A694F.

In an embodiment, there is provided a modified DNA polymerase having an altered characteristic selected from enzyme activity, fidelity, processivity, elongation rate, stability, or solubility, when compared to SEQ ID NO:1 or 2. In an embodiment, the altered characteristic is enzyme activity. In an embodiment, the altered characteristic is fidelity. In an embodiment, the altered characteristic is processivity. In an embodiment, the altered characteristic is elongation rate. In an embodiment, the altered characteristic is stability. In an embodiment, the altered characteristic is solubility. In an embodiment, the altered characteristic is improved strand displacement.

In an embodiment, the altered characteristic is enzyme activity. In an embodiment, the altered characteristic is fidelity. In an embodiment, the altered characteristic is processivity. In an embodiment, the altered characteristic is elongation rate. In an embodiment, the altered characteristic is stability. In an embodiment, the altered characteristic is solubility. In one embodiment, the altered characteristic is an ability to bind and/or incorporate polyphosphate nucleotides, e.g., a tetraphosphate, pentaphosphate, hexaphosphate, heptaphosphate or octophosphate nucleotide.

In some embodiments, the modified polymerase has a k_(cat) that is greater than the parental polymerase. In some embodiments, the modified polymerase has a k_(off) that is less than the parental polymerase. In some embodiments, the modified polymerase has a k_(cat)/k_(off) (i.e., a ratio) that is at least 1.5, 2.0 or 2.5 times greater than the parental polymerase.

In an aspect, there is provided a method of sequencing a nucleic acid template using a variant polymerase as described herein. In an embodiment, the sequencing is with the aid of a nanopore.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to one skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 illustrates an exemplary template used in the displacement assay.

FIG. 2 is a depiction of the k_(off) assay based on fluorescence polarization and an exemplary data trace. Reference is made to Example 3.

FIG. 3 is a graph of representative data from fluorescence polarization based k_(off) assay. Reference is made to Example 4. Shown are traces for a parental polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+Y242A) and a variant of the parental polymerase having one additional mutation (S293M). This is an advanced mutant that was selected based on its extension rate and nucleotide dissociation rate for polyphosphate nucleotides through several generation of mutagenesis on WT Pol6. Using the parental backbone, we looked for improved binding to polyphosphate nucleotide using our standard polarization based k_(off) assay. Shown is an example mutant S293M shows slower rate of nucleotide dissociation compared to the parental polymerase.

FIG. 4 is a graph of a parental polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+Y242A) and two variants of the parental polymerase. Variant 1 further comprises T560M and Variant 2 further comprises K561G relative to the parental polymerase. Reference is made to Example 3. This is an advanced mutant that was selected based on its extension rate and nucleotide dissociation rate for polyphosphate nucleotides through several generation of mutagenesis on WT Pol6. Using the parental backbone, we screened several mutants for its ability to improve extension with polyphosphate nucleotide using our FRET based displacement assay. Shown in the figure is an example of a couple of mutants T560M and K561G, that show increased rate of extension {as reflected by the time to inflection) compared to the parental polymerase.

FIG. 5 is a sequencing trace showing the changes in current that provides a record of the tagged nucleotides as they are incorporated into the growing DNA strand for a parental pol6 (T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+Y242A) showing the changes in current that provides a record of the tagged nucleotides as they are incorporated into the growing DNA strand. Also shown is the template DNA sequence and the called sequence of the nascent strand demonstrating >70% accuracy. Reference is made to Example 5. Example of a sequencing trace with a parental Pol6 {T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+Y242A) showing the changes in current that provides a record of the tagged nucleotides as they are incorporated into the growing DNA strand. Also shown is the template DNA sequence and the called sequence of the nascent strand demonstrating >70% accuracy.

FIG. 6 is a sequencing trace showing the changes in current that provides a record of the tagged nucleotides as they are incorporated into the growing DNA strand for a variant pol6 (T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+Y242A+E585K) derived from the pol6 of FIG. 5. Also shown is the template DNA sequence (SEQ ID NO: 6) and the called sequence (SEQ ID NO: 7) of the nascent strand demonstrating >80% accuracy. Reference is made to Example 5. Example of a sequencing trace with a variant Pol6 (T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+Y242A+E585K) showing the changes in current that provides a record of the tagged nucleotides as they are incorporated into the growing DNA strand. Also shown is the template DNA sequence and the called sequence of the nascent strand demonstrating >80% accuracy.

DETAILED DESCRIPTION

The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Practitioners are particularly directed to Sambrook et al., 1989, and Ausubel F M et al., 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

Numeric ranges are inclusive of the numbers defining the range. The term about is used herein to mean plus or minus ten percent (10%) of a value. For example, “about 100” refers to any number between 90 and 110.

Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

DEFINITIONS

Amino acid: As used herein, the term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and/or substitution with other chemical without adversely affecting their activity. Amino acids may participate in a disulfide bond. The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. It should be noted that all amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus.

Base Pair (bp): As used herein, base pair refers to a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.

Complementary: As used herein, the term “complementary” refers to the broad concept of sequence complementarity between regions of two polynucleotide strands or between two nucleotides through base-pairing. It is known that an adenine nucleotide is capable of forming specific hydrogen bonds (“base pairing”) with a nucleotide which is thymine or uracil. Similarly, it is known that a cytosine nucleotide is capable of base pairing with a guanine nucleotide.

DNA binding affinity: As used herein, the term “DNA-binding affinity” typically refers to the activity of a DNA polymerase in binding DNA nucleic acid. In some embodiments, DNA binding activity can be measured in a two band-shift assay. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3^(rd) ed., Cold Spring Harbor Laboratory Press, N.Y.) at 9.63-9.75 (describing end-labeling of nucleic acids). A reaction mixture is prepared containing at least about 0.5 μg of the polypeptide in about 10 μl of binding buffer (50 mM sodium phosphate buffer (pH 8.0), 10% glycerol, 25 mM KCl, 25 mM MgCl₂). The reaction mixture is heated to 37° C. for 10 min. About 1×10⁴ to 5×10⁴ cpm (or about 0.5-2 ng) of the labeled double-stranded nucleic acid is added to the reaction mixture and incubated for an additional 10 min. The reaction mixture is loaded onto a native polyacrylamide gel in 0.5× Tris-borate buffer. The reaction mixture is subjected to electrophoresis at room temperature. The gel is dried and subjected to autoradiography using standard methods. Any detectable decrease in the mobility of the labeled double-stranded nucleic acid indicates formation of a binding complex between the polypeptide and the double-stranded nucleic acid. Such nucleic acid binding activity may be quantified using standard densitometric methods to measure the amount of radioactivity in the binding complex relative to the total amount of radioactivity in the initial reaction mixture. Other methods of measuring DNA binding affinity are known in the art (see, e.g., Kong et al. (1993) J. Biol. Chem. 268(3):1965-1975).

Elongation rate: As used herein, the term “elongation rate” refers to the average rate at which a DNA polymerase extends a polymer chain. As used herein, a high elongation rate refers to an elongation rate higher than 2 nt/s (e.g., higher than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 nt/s). As used in this application, the terms “elongation rate”, “extension rate” and “incorporation rate” are used interchangeably.

Enzyme activity: As used herein, the term “enzyme activity” refers to the specificity and efficiency of a DNA polymerase. Enzyme activity of a DNA polymerase is also referred to as “polymerase activity,” which typically refers to the activity of a DNA polymerase in catalyzing the template-directed synthesis of a polynucleotide. Enzyme activity of a polymerase can be measured using various techniques and methods known in the art. For example, serial dilutions of polymerase can be prepared in dilution buffer (e.g., 20 mM Tris.Cl, pH 8.0, 50 mM KCl, 0.5% NP 40, and 0.5% Tween-20). For each dilution, 5 μl can be removed and added to 45 μl of a reaction mixture containing 25 mM TAPS (pH 9.25), 50 mM KCl, 2 mM MgCl₂, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP, 0.1 mM dCTP, 12.5 μg activated DNA, 100 μM [α-³²P]dCTP (0.05 μCi/nmol) and sterile deionized water. The reaction mixtures can be incubated at 37° C. (or 74° C. for thermostable DNA polymerases) for 10 minutes and then stopped by immediately cooling the reaction to 4° C. and adding 10 μl of ice-cold 60 mM EDTA. A 25 μl aliquot can be removed from each reaction mixture. Unincorporated radioactively labeled dCTP can be removed from each aliquot by gel filtration (Centri-Sep, Princeton Separations, Adelphia, N.J.). The column eluate can be mixed with scintillation fluid (1 ml). Radioactivity in the column eluate is quantified with a scintillation counter to determine the amount of product synthesized by the polymerase. One unit of polymerase activity can be defined as the amount of polymerase necessary to synthesize 10 nmole of product in 30 minutes (Lawyer et al. (1989) J. Biol. Chem. 264:6427-647). Other methods of measuring polymerase activity are known in the art (see, e.g. Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, N.Y.)).

Purified: As used herein, “purified” means that a molecule is present in a sample at a concentration of at least 90% by weight, or at least 95% by weight, or at least 98% by weight of the sample in which it is contained.

Isolated: An “isolated” molecule is a nucleic acid molecule that is separated from at least one other molecule with which it is ordinarily associated, for example, in its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the nucleic acid molecule, but the nucleic acid molecule is present extrachromasomally or at a chromosomal location that is different from its natural chromosomal location.

% homology: The term “% homology” is used interchangeably herein with the term “% identity” herein and refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence that encodes any one of the inventive polypeptides or the inventive polypeptide's amino acid sequence, when aligned using a sequence alignment program.

For example, as used herein, 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence. Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., the coding sequence for any one of the inventive polypeptides, as described herein.

Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul, et al., 1990 and Altschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTN and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402, 1997.)

A preferred alignment of selected sequences in order to determine “% identity” between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

Modified DNA polymerase: As used herein, the term “modified DNA polymerase” refers to a DNA polymerase originated from another (i.e., parental) DNA polymerase and contains one or more amino acid alterations (e.g., amino acid substitution, deletion, or insertion) compared to the parental DNA polymerase. In some embodiments, a modified DNA polymerases of the invention is originated or modified from a naturally-occurring or wild-type DNA polymerase. In some embodiments, a modified DNA polymerase of the invention is originated or modified from a recombinant or engineered DNA polymerase including, but not limited to, chimeric DNA polymerase, fusion DNA polymerase or another modified DNA polymerase. Typically, a modified DNA polymerase has at least one changed phenotype compared to the parental polymerase.

Mutation: As used herein, the term “mutation” refers to a change introduced into a parental sequence, including, but not limited to, substitutions, insertions, deletions (including truncations). The consequences of a mutation include, but are not limited to, the creation of a new character, property, function, phenotype or trait not found in the protein encoded by the parental sequence.

Mutant: As used herein, the term “mutant” refers to a modified protein which displays altered characteristics when compared to the parental protein. The terms “variant” and “mutant” are used interchangeably herein.

Wild-type: As used herein, the term “wild-type” refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally-occurring source.

Fidelity: As used herein, the term “fidelity” refers to either the accuracy of DNA polymerization by template-dependent DNA polymerase or the measured difference in k_(off) of the correct nucleotide vs incorrect nucleotide binding to the template DNA. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an inaccurate nucleotide, i.e., a nucleotide that is not incorporated at a template-dependent manner). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3′→5′ exonuclease activity of a DNA polymerase. The term “high fidelity” refers to an error rate less than 4.45×10 ⁻⁶(e.g., less than 4.0×10⁻⁶, 3.5×10⁻⁶, 3.0×10⁻⁶, 2.5×10⁻⁶, 2.0×10⁻⁶, 1.5×10⁻⁶, 1.0×10⁻⁶, 0.5×10⁻⁶) mutations/nt/doubling. The fidelity or error rate of a DNA polymerase may be measured using assays known to the art. For example, the error rates of DNA polymerases can be tested as described herein or as described in Johnson, et al., Biochim Biophys Acta. 2010 May; 1804(5): 1041-1048.

Nanopore: The term “nanopore,” as used herein, generally refers to a pore, channel or passage formed or otherwise provided in a membrane. A membrane may be an organic membrane, such as a lipid bilayer, or a synthetic membrane, such as a membrane formed of a polymeric material. The membrane may be a polymeric material. The nanopore may be disposed adjacent or in proximity to a sensing circuit or an electrode coupled to a sensing circuit, such as, for example, a complementary metal-oxide semiconductor (CMOS) or field effect transistor (FET) circuit. In some examples, a nanopore has a characteristic width or diameter on the order of 0.1 nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alpha-hemolysin, MspA are examples of a protein nanopore.

Nucleotide: As used herein, a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.

The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3′ or 5′ position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a “base sequence” or “nucleotide sequence,” and is represented herein by a formula whose left to right orientation is in the conventional direction of 5′-terminus to 3′-terminus. As used herein, a “modified nucleotide” refers to a polyphosphate, e.g., 3, 4, 5, 6, 7 or 8 phosphates, nucleotide.

Oligonucleotide or Polynucleotide: As used herein, the term “oligonucleotide” is defined as a molecule including two or more deoxyribonucleotides and/or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be derived synthetically or by cloning. As used herein, the term “polynucleotide” refers to a polymer molecule composed of nucleotide monomers covalently bonded in a chain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.

Polymerase: As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotide (i.e., the polymerase activity).

Generally, the enzyme will initiate synthesis at the 3′-end of the primer annealed to a polynucleotide template sequence, and will proceed toward the 5′ end of the template strand. A “DNA polymerase” catalyzes the polymerization of deoxynucleotides.

Primer: As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, e.g., in the presence of four different nucleotide triphosphates and thermostable enzyme in an appropriate buffer (“buffer” includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. The primer is preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the thermostable enzyme. The exact lengths of the primers will depend on many factors, including temperature, source of primer and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 nucleotides, although it may contain more or few nucleotides. Short primer molecules generally require colder temperatures to form sufficiently stable hybrid complexes with template.

Processivity: As used herein, “processivity” refers to the ability of a polymerase to remain attached to the template and perform multiple modification reactions. “Modification reactions” include but are not limited to polymerization, and exonucleolytic cleavage. In some embodiments, “processivity” refers to the ability of a DNA polymerase to perform a sequence of polymerization steps without intervening dissociation of the enzyme from the growing DNA chains. Typically, “processivity” of a DNA polymerase is measured by the length of nucleotides (for example 20 nts, 300 nts, 0.5-1 kb, or more) that are polymerized or modified without intervening dissociation of the DNA polymerase from the growing DNA chain. “Processivity” can depend on the nature of the polymerase, the sequence of a DNA template, and reaction conditions, for example, salt concentration, temperature or the presence of specific proteins. As used herein, the term “high processivity” refers to a processivity higher than 20 nts (e.g., higher than 40 nts, 60 nts, 80 nts, 100 nts, 120 nts, 140 nts, 160 nts, 180 nts, 200 nts, 220 nts, 240 nts, 260 nts, 280 nts, 300 nts, 320 nts, 340 nts, 360 nts, 380 nts, 400 nts, or higher) per association/disassociation with the template. Processivity can be measured according the methods defined herein and in WO 01/92501 A1 (MJ Bioworks, Inc., Improved Nucleic Acid Modifying Enzymes, published 6 Dec. 2001).

Synthesis: As used herein, the term “synthesis” refers to any in vitro method for making new strand of polynucleotide or elongating existing polynucleotide (i.e., DNA or RNA) in a template dependent manner Synthesis, according to the invention, includes amplification, which increases the number of copies of a polynucleotide template sequence with the use of a polymerase. Polynucleotide synthesis (e.g., amplification) results in the incorporation of nucleotides into a polynucleotide (i.e., a primer), thereby forming a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules. “DNA synthesis,” as used herein, includes, but is not limited to, PCR, the labeling of polynucleotide (i.e., for probes and oligonucleotide primers), polynucleotide sequencing.

Template DNA molecule: As used herein, the term “template DNA molecule” refers to a strand of a nucleic acid from which a complementary nucleic acid strand is synthesized by a DNA polymerase, for example, in a primer extension reaction.

Template-dependent manner: As used herein, the term “template-dependent manner” refers to a process that involves the template dependent extension of a primer molecule (e.g., DNA synthesis by DNA polymerase). The term “template-dependent manner” typically refers to polynucleotide synthesis of RNA or DNA wherein the sequence of the newly synthesized strand of polynucleotide is dictated by the well-known rules of complementary base pairing (see, for example, Watson, J. D. et al., In: Molecular Biology of the Gene, 4th Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1987)).

Tag: As used herein, the term “tag” refers to a detectable moiety that may be atoms or molecules, or a collection of atoms or molecules. A tag may provide an optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature, which signature may be detected with the aid of a nanopore.

Tagged Nucleotide: As used herein, the term “tagged nucleotide” refers to a nucleotide or modified nucleotide that has a tag attached. The tag may be attached covalently to the sugar, the phosphate (or polyphosphate) or base. The tag may be on the terminal phosphate.

Vector: As used herein, the term “vector” refers to a nucleic acid construct designed for transfer between different host cells. An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

The polymerase variants provided for herein are useful in the chip-based polynucleotide sequencing as described in WO2013/188841 (Genia Technologies, Inc., Chip Set-Up and High-Accuracy Nucleic Acid Sequencing, published 19 Dec. 2013).

Desired characteristics of a polymerase that finds use in sequencing DNA are:

-   -   a. Slow k_(off) (for modified nucleotide)     -   b. Fast k_(on) (for modified nucleotide)     -   c. High fidelity     -   d. Low exonuclease activity     -   e. DNA strand displacement     -   f. Faster k_(cat) (for modified nucleotide substrates)     -   g. Increased stability     -   h. Processivity     -   i. Salt tolerance     -   j. Compatible with attachment to nanopore     -   k. Ability to incorporate a polyphosphates having 4, 5, 6, 7 or         8 phosphates, e.g., quadraphosphate, pentaphosphate,         hexaphosphate, heptaphosphate or octophosphate nucleotide     -   l. Sequencing accuracy     -   m. Long read lengths, i.e., long continuous reads.         Nomenclature

In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used.

For ease of reference, polymerase variants of the application are described by use of the following nomenclature:

Original amino acid(s): position(s): substituted amino acid(s). According to this nomenclature, for instance the substitution of serine by an alanine in position 242 is shown as:

-   -   Ser242Ala or S242A

Multiple mutations are separated by plus signs, i.e.:

-   -   Ala30Asp+Glu34Ser or A30N+E34S         representing mutations in positions 30 and 34 substituting         alanine and glutamic acid for asparagine and serine,         respectively.

When one or more alternative amino acid residues may be inserted in a given position it is indicated as: A30N/E or A30N or A30E.

Unless otherwise stated, the number of the residues corresponds to the residue numbering of SEQ ID NO:2.

Site-Directed Mutagenesis of Polymerase

Clostridium phage phiCPV4 wild type sequences are provided herein (SEQ ID NO:3, nucleic acid coding region plus a His-tag; SEQ ID NO:1, protein coding region) and available elsewhere (National Center for Bioinformatics or GenBank Accession Numbers AFH27113).

Point mutations may be introduced using QuikChange Lightning 2 kit (Stategene/Agilent) following manufacturer's instructions.

Primers can be ordered from commercial companies, e.g., IDT DNA.

Nanopore Assembly and Insertion

The methods described herein can use a nanopore having a polymerase attached to the nanopore. In some cases, it is desirable to have one and only one polymerase per nanopore (e.g., so that only one nucleic acid molecule is sequenced at each nanopore). However, many nanopores, including, e.g., alpha-hemolysin (aHL), can be multimeric proteins having a plurality of subunits (e.g., 7 subunits for aHL). The subunits can be identical copies of the same polypeptide. Provided herein are multimeric proteins (e.g., nanopores) having a defined ratio of modified subunits (e.g., a-HL variants) to un-modified subunits (e.g., a-HL). Also provided herein are methods for producing multimeric proteins (e.g., nanopores) having a defined ratio of modified subunits to un-modified subunits.

With reference to FIG. 27 of WO2014/074727 (Genia Technologies, Inc.), a method for assembling a protein having a plurality of subunits comprises providing a plurality of first subunits 2705 and providing a plurality of second subunits 2710, where the second subunits are modified when compared with the first subunits. In some cases, the first subunits are wild-type (e.g., purified from native sources or produced recombinantly). The second subunits can be modified in any suitable way. In some cases, the second subunits have a protein (e.g., a polymerase) attached (e.g., as a fusion protein).

The modified subunits can comprise a chemically reactive moiety (e.g., an azide or an alkyne group suitable for forming a linkage). In some cases, the method further comprises performing a reaction (e.g., a Click chemistry cycloaddition) to attach an entity (e.g., a polymerase) to the chemically reactive moiety.

The method can further comprise contacting the first subunits with the second subunits 2715 in a first ratio to form a plurality of proteins 2720 having the first subunits and the second subunits. For example, one part modified aHL subunits having a reactive group suitable for attaching a polymerase can be mixed with six parts wild-type aHL subunits (i.e., with the first ratio being 1:6). The plurality of proteins can have a plurality of ratios of the first subunits to the second subunits. For example, the mixed subunits can form several nanopores having a distribution of stoichiometries of modified to un-modified subunits (e.g., 1:6, 2:5, 3:4).

In some cases, the proteins are formed by simply mixing the subunits. In the case of aHL nanopores for example, a detergent (e.g., deoxycholic acid) can trigger the aHL monomer to adopt the pore conformation. The nanopores can also be formed using a lipid (e.g., 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DoPhPC)) and moderate temperature (e.g., less than about 100° C.). In some cases, mixing DPhPC with a buffer solution creates large multi-lamellar vesicles (LMV), and adding aHL subunits to this solution and incubating the mixture at 40° C. for 30 minutes results in pore formation.

If two different types of subunits are used (e.g., the natural wild type protein and a second aHL monomer which can contain a single point mutation), the resulting proteins can have a mixed stoichiometry (e.g., of the wild type and mutant proteins). The stoichiometry of these proteins can follow a formula which is dependent upon the ratio of the concentrations of the two proteins used in the pore forming reaction. This formula is as follows: 100P _(m)=100[n!/m!(n−m)!]·f _(mut) ^(m) ·f _(wt) ^(n˜m), where

-   -   P_(m)=probability of a pore having m number of mutant subunits     -   n=total number of subunits (e.g., 7 for aHL)     -   m=number of “mutant” subunits     -   f_(mut)=fraction or ratio of mutant subunits mixed together     -   f_(wt)=fraction or ratio of wild-type subunits mixed together

The method can further comprise fractionating the plurality of proteins to enrich proteins that have a second ratio of the first subunits to the second subunits 2725. For example, nanopore proteins can be isolated that have one and only one modified subunit (e.g., a second ratio of 1:6). However, any second ratio is suitable. A distribution of second ratios can also be fractionated such as enriching proteins that have either one or two modified subunits. The total number of subunits forming the protein is not always 7 (e.g., a different nanopore can be used or an alpha-hemolysin nanopore can form having six subunits) as depicted in FIG. 27 of WO2014/074727. In some cases, proteins having only one modified subunit are enriched. In such cases, the second ratio is 1 second subunit per (n−1) first subunits where n is the number of subunits comprising the protein.

The first ratio can be the same as the second ratio, however this is not required. In some cases, proteins having mutated monomers can form less efficiently than those not having mutated subunits. If this is the case, the first ratio can be greater than the second ratio (e.g., if a second ratio of 1 mutated to 6 non-mutated subunits are desired in a nanopore, forming a suitable number of 1:6 proteins may require mixing the subunits at a ratio greater than 1:6).

Proteins having different second ratios of subunits can behave differently (e.g., have different retention times) in a separation. In some cases, the proteins are fractionated using chromatography, such as ion exchange chromatography or affinity chromatography. Since the first and second subunits can be identical apart from the modification, the number of modifications on the protein can serve as a basis for separation. In some cases, either the first or second subunits have a purification tag (e.g., in addition to the modification) to allow or improve the efficiency of the fractionation. In some cases, a poly-histidine tag (His-tag), a streptavidin tag (Strep-tag), or other peptide tag is used. In some instances, the first and second subunits each comprise different tags and the fractionation step fractionates on the basis of each tag. In the case of a His-tag, a charge is created on the tag at low pH (Histidine residues become positively charged below the pKa of the side chain). With a significant difference in charge on one of the aHL molecules compared to the others, ion exchange chromatography can be used to separate the oligomers which have 0, 1, 2, 3, 4, 5, 6, or 7 of the “charge-tagged” aHL subunits. In principle, this charge tag can be a string of any amino acids which carry a uniform charge. FIG. 28 and FIG. 29 show examples of fractionation of nanopores based on a His-tag. FIG. 28 shows a plot of ultraviolet absorbance at 280 nanometers, ultraviolet absorbance at 260 nanometers, and conductivity. The peaks correspond to nanopores with various ratios of modified and unmodified subunits. FIG. 29 of WO2014/074727 shows fractionation of aHL nanopores and mutants thereof using both His-tag and Strep-tags.

In some cases, an entity (e.g., a polymerase) is attached to the protein following fractionation. The protein can be a nanopore and the entity can be a polymerase. In some instances, the method further comprises inserting the proteins having the second ratio subunits into a bilayer.

In some situations, a nanopore can comprise a plurality of subunits. A polymerase can be attached to one of the subunits and at least one and less than all of the subunits comprise a first purification tag. In some examples, the nanopore is alpha-hemolysin or a variant thereof. In some instances, all of the subunits comprise a first purification tag or a second purification tag. The first purification tag can be a poly-histidine tag (e.g., on the subunit having the polymerase attached).

Polymerase Attached to Nanopore

In some cases, a polymerase (e.g., DNA polymerase) is attached to and/or is located in proximity to the nanopore. The polymerase can be attached to the nanopore before or after the nanopore is incorporated into the membrane. In some instances, the nanopore and polymerase are a fusion protein (i.e., single polypeptide chain).

The polymerase can be attached to the nanopore in any suitable way. In some cases, the polymerase is attached to the nanopore (e.g., hemolysin) protein monomer and then the full nanopore heptamer is assembled (e.g., in a ratio of one monomer with an attached polymerase to 6 nanopore (e.g., hemolysin) monomers without an attached polymerase). The nanopore heptamer can then be inserted into the membrane.

Another method for attaching a polymerase to a nanopore involves attaching a linker molecule to a hemolysin monomer or mutating a hemolysin monomer to have an attachment site and then assembling the full nanopore heptamer (e.g., at a ratio of one monomer with linker and/or attachment site to 6 hemolysin monomers with no linker and/or attachment site). A polymerase can then be attached to the attachment site or attachment linker (e.g., in bulk, before inserting into the membrane). The polymerase can also be attached to the attachment site or attachment linker after the (e.g., heptamer) nanopore is formed in the membrane. In some cases, a plurality of nanopore-polymerase pairs are inserted into a plurality of membranes (e.g., disposed over the wells and/or electrodes) of the biochip. In some instances, the attachment of the polymerase to the nanopore complex occurs on the biochip above each electrode.

The polymerase can be attached to the nanopore with any suitable chemistry (e.g., covalent bond and/or linker). In some cases, the polymerase is attached to the nanopore with molecular staples. In some instances, molecular staples comprise three amino acid sequences (denoted linkers A, B and C). Linker A can extend from a hemolysin monomer, Linker B can extend from the polymerase, and Linker C then can bind Linkers A and B (e.g., by wrapping around both Linkers A and B) and thus the polymerase to the nanopore. Linker C can also be constructed to be part of Linker A or Linker B, thus reducing the number of linker molecules.

In some instances, the polymerase is linked to the nanopore using Solulink™ chemistry. Solulink™ can be a reaction between HyNic (6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB (4-formylbenzoate, an aromatic aldehyde). In some instances, the polymerase is linked to the nanopore using Click chemistry (available from LifeTechnologies for example). In some cases, zinc finger mutations are introduced into the hemolysin molecule and then a molecule is used (e.g., a DNA intermediate molecule) to link the polymerase to the zinc finger sites on the hemolysin.

Other linkers that may find use in attaching the polymerase to a nanopore are direct genetic linkage (e.g., (GGGGS)₁₋₃ (SEQ ID NO: 4) amino acid linker), transglutaminase mediated linking (e.g., RSKLG (SEQ ID NO: 5)), sortase mediated linking, and chemical linking through cysteine modifications. Specific linkers contemplated as useful herein are (GGGGS)₁₋₃ (SEQ ID NO: 4), K-tag (RSKLG (SEQ ID NO: 5)) on N-terminus, ΔTEV site (12-25), ΔTEV site+N-terminus of SpyCatcher (12-49).

Apparatus Set-Up

The nanopore may be formed or otherwise embedded in a membrane disposed adjacent to a sensing electrode of a sensing circuit, such as an integrated circuit. The integrated circuit may be an application specific integrated circuit (ASIC). In some examples, the integrated circuit is a field effect transistor or a complementary metal-oxide semiconductor (CMOS). The sensing circuit may be situated in a chip or other device having the nanopore, or off of the chip or device, such as in an off-chip configuration. The semiconductor can be any semiconductor, including, without limitation, Group IV (e.g., silicon) and Group III-V semiconductors (e.g., gallium arsenide). See, for example, WO 2013/123450, for the apparatus and device set-up for sensing a nucleotide or tag.

Pore based sensors (e.g., biochips) can be used for electro-interrogation of single molecules. A pore based sensor can include a nanopore of the present disclosure formed in a membrane that is disposed adjacent or in proximity to a sensing electrode. The sensor can include a counter electrode. The membrane includes a trans side (i.e., side facing the sensing electrode) and a cis side (i.e., side facing the counter electrode).

In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds).

EXAMPLES

The present invention is described in further explained in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 Directed Mutagenesis

This example illustrates the introduction of a mutation into a pol6 polymerase at a desired position.

DNA encoding the His-tagged wild-type pol6 was purchased from a commercial source (DNA 2.0, Menlo Park, Calif.). The sequence was verified by sequencing.

For the mutant screen, we expressed the polymerase as is (N-ter His-Pol6). In order to test the pol hits on the chip, we engineered in a SpyCatcher domain in N-ter or C-ter of Pol6.

Rational positions to impact Pol6-nucleotide binding were identified based on homology modeling of known crystal structures.

For the primary screen, each of the rational positions were mutated into Gly, Ala, Leu, Glu, Gin, Lys, Arg, Tyr, Phe, Trp, Thr or Met using the Q5 mutagenesis protocol. Subsequent screens mutated positions to all other 19 amino acid possibilities.

The primers for each mutagenesis reaction was designed using the NEB base changer protocol and ordered in 96-well plate format from IDT.

The forward and reverse primers were 5′ phosphorylated in high throughput (HTP) format using the T4 polynucleotidekinase (PNK) purchased from NEB. A typical 25-μl reaction contained 15 μl of primer at 10 μM, 5 μl of 5× reaction buffer (from NEB), 1.25 μl PNK enzyme, 3.75 μl water. The reaction was performed at 37° C. for 30 min and the enzyme heat inactivated at 65° C. for 20 min.

PCR mutagenesis was performed using Q5 DNA polymerase from NEB. A typical 25 μl reaction contained 5 μl of Q5 buffer, 5 μl of GC enhancer, 0.5 ul of 10 mM dNTPs, 1.25 μl of 10 μM phosphorylated mutagenesis primers forward and reverse, 0.25 μl Q5 polymerase and 1 μl of 5 ng/ml wild type Pol6 template, i.e., His-Pol6, and 10.75 μl H₂0.

Once PCR is complete, 0.5 μl of Dpn1 was added to 25 μl PCR mix and incubated at 37° C. for 1 hr.

Add 2.5 μl of Dpn1 treated PCR product with 2.5 μl of Blunt/TA ligase master mix. Incubate at room temperature for 1 hr.

Add 1 μl of ligation mix to 20 ul of 96-well BL21 DE3 cells (EMD Millipore) and incubate on ice for 5 min.

Heat shock at 42° C. for exactly 30 sec using the PCR device and place on ice for 2 min.

Add 80 μl of SOC and incubate at 37° C. incubator for 1 hr without shaking.

Add 100 μl of SOC or ultra pure water and plate them in 48-well LB-agar plates with 50-100 μg/ml kanamycin.

Example 2 Expression and Purification

The following example details how the pol6 variants were expressed and purified using a high throughput method.

DNA encoding the variants in the pD441 vector (expression plasmid) was transformed into competent E. coli and glycerol stocks made. Starting from a tiny pick of the glycerol stock, grow 1 ml starter culture in LB with 0.2% Glucose and 100 μg/ml Kanamycin for approximately 8 hrs. Transfer 25 μl of log phase starter culture into 1 ml of expression media (Terrific Broth (TB) autoinduction media supplemented with 0.2% glucose, 50 mM Potassium Phosphate, 5 mM MgCl₂ and 100 μg/ml Kanamycin) in 96-deep well plates. The plates were incubated with shaking at 250-300 rpm for 36-40 hrs at 28° C.

Cells were then harvested via centrifugation at 3200×g for 30 minutes at 4° C. The media was decanted off and the cell pellet resuspended in 200 μl pre-chilled lysis buffer (20 mM Potassium Phosphate pH 7.5, 100 mM NaCl, 0.5% Tween20, 5 mM TCEP, 10 mM Imidazole, 1 mM PMSF, 1× Bug Buster, 100 μg/ml Lysozyme and protease inhibitors) and incubate at room temperature for 20 min with mild agitation. Then add 20 μl from a 10 Ox stock to a final concentration of 100 μg/ml DNase, 5 mM MgCl2, 100 μg/ml RNase I and incubate in on ice for 5-10 min to produce a lysate. Supplement the lysate with 200 μl of 1M Potassium Phosphate, pH 7.5 (Final concentration will be about 0.5M Potassium phosphate in 400 μl lysate) and filter through Pall filter plates (Part #5053, 3 micron filters) via centrifugation at approximately 1500 rpm at 4 C for 10 minutes. The clarified lysates were then applied to equilibrated 96-well His-Pur Cobalt plates (Pierce Part#90095) and bind for 15-30 min.

The flow through (FT) was collected by centrifugation at 500×G for 3 min. The FT was then washed 3 times with 400 ul of wash buffer 1 (0.5M Potassium Phosphate pH 7.5, 1M NaCl 5 mM TCEP, 20 mM Imidazole+0.5% Tween20). The FT was then washed twice in 400 ul wash buffer 2 (50 mM Tris pH 7.4, 200 mM KCl, 5 mM TCEP, 0.5% Tween20, 20 mM Imidazole).

The Pol6 was eluted using 200 μl elution buffer (50 mM Tris Ph7.4, 200 mM KCl, 5 mM TCEP, 0.5% Tween20, 300 mM Imidazole, 25% Glycerol) and collected after 1-2 min incubation. Reapply eluate to the same His-Pur plate 2-3 times to get concentrated Pol6 in elute. The purified polymerase is >95% pure as evaluated by SDS-PAGE. The protein concentration is ˜3 uM (0.35 mg/ml) with a 260/280 ratio of 0.6 as evaluated by Nanodrop.

Polymerase activity is checked by FRET based extension assay (see Example 3).

Example 3 Fluorescence Resonance Energy Transfer (FRET) Based Extension Assay

The effect of mutations of amino acids of the variant polymerases on their ability to incorporate polyphosphate nucleotide nucleotides at high salt conditions was tested with a FRET based extension assay.

For this assay, the fluorophore was Alexa/Atto 488 and the quencher was Iowa black.

The sequences used were as follows:

(SEQ ID NO: 6) 5-/5ATTO488N/AGA GTG ATA GTA TGA TTA TGT AGA TGT AGG ATT TGA TAT GTG AGT AGC CGA ATG AAA CCT TTG GTT TCA TTC GG-3 (SEQ ID NO: 7) 5-TTT TCA TAA TCA TAC TAT CAC TCT/3IABkFQ/-3

The polymerase was pre-bound to the hairpin template in low 75 mM KGlu, and at time=0 Mg2+ was injected to initiate extension of the template, which displaced the quencher strand in the process, resulting in increased fluorescence. Fluorescence was monitored using a BMG labtech fluorescence plate reader.

The Final buffer composition used in the assay was as described in Table 1.

TABLE 1 Final Buffer Composition. Reagent [Final] NaCl/KGlu 150 or 300 mM HEPES 7.5 25 mM EDTA 0.2 mM TritonX-100 0.05 % TCEP 5 mM BSA 25 ug/ml Template 50 nM dA, C, G, T 10 uM MgCl 5 mM

The extension curves for an exemplary parental polymerase, and variants thereof are shown in FIG. 4, respectively.

The data show that variant polymerase Pol6 comprising mutations display a decrease in the rate of dissociation from the DNA template, and therefore increased processivity. Accordingly, these variant polymerases are also expected to have increased sequencing lifetime, and to increase the length of the reads during sequencing events.

Example 4 Determination of K_(OFF)

This example provides an alternative method using fluorescence polarization for determining the k_(off).

An assay buffer comprising 25 mM Tris pH7.0, 75 mM KCl, 0.01% Triton-X100, 1×BSA (100 ug/ml), 0.5 mM EDTA, 2 mM CaCl2, 2 mM DTT, was used to prepare an assay master mix containing 250 nM hairpin fluorescein-labeled DNA template and 250 nM dC6P-C6-Cy3 tagged nucleotide. Fifty five microliters of the master mix were added to each of the wells of a black 96-well costar plate; and 20 μl of polymerase mutants, which had been purified from 1 ml cultures, were added in a high throughput (HTP) format. The plate was shaken on a plate shaker for 1 minute to allow for the formation of homogenous ternary complexes of polymerase-DNA template-nucleotide. The plate was placed in a BMG polarstar plate reader (BMG LABTECH Inc., N.C.) and target millipolarization was adjusted to 200 mP and 10% to have a gain around 2000. The excitation filter was set to 485 nM and the emission filter was set to 590-20 nM. The injector was primed with 1 ml of 1 mM dCTP chaser nucleotide solution. Data was collected with minimum 30 flashes per well per interval and 60 sec total read time for the start. The flashes were increased to 50 or higher and longer read times taken for the hit mutants that showed slow dissociation. Data collection began with the injection of 25 μl of 1 mM dCTP.

See FIG. 3 for representative data from fluorescence polarization based k_(off) assay for variant parental polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+Y242A) and a variant polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+Y242A+S293M). mP is millipolarization. Preformed ternary complex of polymerase-DNA template—dCnP-Alexa555 is chased with native dCTP and polarization dCnP-Alexa555 was monitored over time.

Example 5 Attachment to Nanopore

This example provides methods of attaching a variant polymerase to a nanopore, e.g., α-hemolysin.

The polymerase may be coupled to the nanopore by any suitable means. See, for example, PCT/US2013/068967 (published as WO2014/074727; Genia Technologies, Inc.), PCT/US2005/009702 (published as WO2006/028508; President and Fellows of Harvard College), and PCT/US2011/065640 (published as WO2012/083249; Columbia University).

The polymerase, e.g., a variant pol6 DNA Polymerase, was coupled to a protein nanopore (e.g. alpha-hemolysin), through a linker molecule. Specifically, the SpyTag and SpyCatcher system, that spontaneously forms covalent isopeptide linkages under physiological conditions was used. See, for example, Li et al, J Mol Biol. 2014 Jan. 23; 426(2):309-17.

The pol6 variant SpyCatcher HisTag was expressed according to Example 2 and purified using a cobalt affinity column. The SpyCatcher polymerase and the SpyTag oligomerized nanopore protein were incubated overnight at 4° C. in 3 mM SrCl₂. The 1:6-polymerase-template complex was then purified using size-exclusion chromatography.

The linker was attached at either the N-terminal or C-terminal of the pol6 variant. The N-terminally attached variants were found to be more robust, e.g., more stable. Therefore, N-terminally attached linkers were used.

Example 6 Sequencing Template DNA Using Tagged Nucleotides

This example demonstrates that the variant polymerase is functional in a sequencing by synthesis method on a biochip.

AC sequencing of a template using a parental Pol6 polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+K561 G+K541R+T544A+Y242A) and a variant Pol6 polymerase (T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+Y242A+E585K) at 20 mM Hepes, pH 8, 500 mM Potassium Glutamate and 5 mM MgCl2 at room temperature. A nanopore with attached polymerase was embedded in the lipid bilayer as described herein. Primed DNA was added and allowed to complex with the polymerase. Four different tagged nucleotides were added at a concentration of 20 μM. The sequencing by synthesis may proceed as described in WO 2014/074727 entitled “Nucleic Acid Sequencing Using Tags.” The trace for the parental polymerase is shown in FIG. 5 and indicates a sequencing accuracy of 71%. The trace in FIG. 6 shows a sequencing accuracy of 81% for a heteropolymer template and 96 bp read length for the variant polymerase.

Example 7 Temperature Assay

The effect of mutations of amino acids of the variant polymerases on their ability to incorporate polyphosphate nucleotide nucleotides at high temperatures was tested with a FRET based extension assay. High temperatures were defined as temperatures higher than the melting point of the wild type enzyme, or the temperature at which the wild type enzyme was denatured.

For this assay, the fluorophore was Alexa/Atto 488 and the quencher was Iowa black.

The sequences used were as follows:

(SEQ ID NO: 6) 5-/5ATTO488N/AGA GTG ATA GTA TGA TTA TGT AGA TGT AGG ATT TGA TAT GTG AGT AGC CGA ATG AAA CCT TTG GTT TCA TTC GG-3 (SEQ ID NO: 7) 5-TTT TCA TAA TCA TAC TAT CAC TCT/3IABkFQ/-3

The polymerase was pre-bound to the hairpin template in low 75 mM KGlu, and incubated at 45° C. for 30 minutes. Then the FRET based extension assay was run as usual: at time=0 Mg2+ was injected to initiate extension of the template, which displaced the quencher strand in the process, resulting in increased fluorescence. Fluorescence was monitored using a BMG labtech fluorescence plate reader.

The Final buffer composition used in the assay was as described in Table 2:

TABLE 2 Final Buffer Composition. Reagent [Final] NaCl/KGlu 150 or 300 mM HEPES 7.5 25 mM EDTA 0.2 mM TritonX-100 0.05 % TCEP 5 mM BSA 25 ug/ml Template 50 nM dA, C, G, T 10 uM MgCl 5 mM

The fold improvement of various mutations compared to control are shown in Table 3.

TABLE 3 Fold Improvement of Mutations Compared to Control. Mutations Backbone Kext Koff Kon Temperature K655Y/K682Y 121X2P1 0.63 14.56 0.94 0.89 Q221W/K655G 121X2P1 1.7 10.05 1.18 0.49 Q221W/V270A/K655G 121X2P1 1.62 7.36 1.31 0.24 N642E/K655Y 121X2P1 0.59 6.19 0.77 0.59 I652E 121X2P1 0.64 6.18 0.77 0.97 T651M/K655A 121X2P1 0.55 6.17 0.43 1.09 I652E/V658F/F483L 121X2P1 0.79 5.92 0.79 0.58 I652E/V658F 121X2P1 0.73 4.77 0.81 0.98 I628M/T651M/K655A 121X2P1 0.92 4.47 1.08 0.92 E590R/N642E/K655Y 121X2P1 0.58 4.37 0.73 0.05 I652E/K655Y 121X2P1 0.55 3.97 0.97 1.05 L549R/D684M 121X2P1 0.68 3.76 1.5 0.75 N298K/G553A 121X2P1 0.64 3.68 0.61 0.63 Q221W/I638E 121X2P1 1.39 3.67 0.84 0.62 K655Y 121X2P1 0.74 3.66 0.91 Q221W 121X2P1 1.32 3.46 1.31 0.57 T560F/V634L 121X2P1 0.8 3.45 0.9 0 I638A/I652E 121X2P1 0.77 3 0.45 0.69 Q221W/F732Y 121X2P1 1.57 2.73 1.45 0.59 I652Q/H227N 121X2P1 0.81 2.58 n.d 0.99 G588E/I652Q 121X2P1 0.7 2.31 0.99 0.04 S294Y 121X2P1 0.64 2.21 0.97 0.25 G553A 121X2P1 0.81 2.15 0.74 0.85 K682F 121X2P1 0.74 2.14 0.84 0.77 G062E/L361M 121X2P1 0.79 1.98 0.83 0.49 L361M/T560Y 121X2P1 1 1.96 0.92 0 I652Q 121X2P1 0.91 1.92 0.81 1.29 F558M 121X2P1 0.68 1.9 1.14 0.31 L471Y/L549R 121X2P1 1.05 1.87 1.14 0.8 S293Q/L653M 121X2P1 1.21 1.76 3.09 0.21 Y629W/K655G 121X2P1 1.2 1.72 1.29 1.37 G062E 121X2P1 0.79 1.71 1.02 0.39 S293Q/S294Y/I638E 121X2P1 0.77 1.71 1.04 0.07 I652G 121X2P1 1.1 1.66 1.07 0.56 V270A/V634L 121X2P1 1.04 1.65 0.9 0.68 T560Y 121X2P1 0.88 1.63 1.05 0.06 L653M 121X2P1 0.73 1.63 1.18 1.01 S293Q/S294Y/I638E/W479L 121X2P1 1.07 1.55 1.4 0.08 Y414K 121X2P1 0.85 1.49 0.96 0.95 F521M 121X2P1 0.97 1.44 1.07 0.7 K235F 121X2P1 0.66 1.42 0.65 0.88 Y414I 121X2P1 0.89 1.39 1.12 1.19 I638E 121X2P1 1 1.35 0.85 0.88 L361M 121X2P1 1 1.34 0.7 0.65 Y629W 121X2P1 0.95 1.33 1.32 0.9 I628M 121X2P1 1.09 1.31 1.12 0.61 P523K 121X2P1 0.86 1.31 0.85 0.81 V270A 121X2P1 0.91 1.3 0.85 0.88 K682M/I698L 121X2P1 0.64 1.27 0.79 1.02 G553A/A554Q/F732Y 121X2P1 1.28 1.26 1.68 0 L538R 121X2P1 1.02 1.25 1.06 0.89 M738W 121X2P1 1.02 1.25 0.98 0.6 C688A 121X2P1 0.75 1.25 1.14 1.1 K374H/T651M 121X2P1 1.02 1.24 0.98 0.44 N535L 121X2P1 0.6 1.24 2.08 0.44 E502P/C688A 121X2P1 0.73 1.24 1.13 1.19 T464P/C688A 121X2P1 0.76 1.21 nd 1.28 I628M/A643R 121X2P1 1.11 1.19 1.05 0.83 K235F/L708H 121X2P1 0.61 1.19 0.6 0.83 I722R/C688A 121X2P1 0.73 1.18 0.87 1.44 K682V/I698L 121X2P1 0.63 1.17 0.76 1.21 I698L 121X2P1 0.74 1.16 0.9 1.39 I722R/E502P 121X2P1 0.92 1.15 0.85 0.9 S294T/I628M/A643R 121X2P1 1.32 1.12 1.33 0.22 L361M/I638A/K655G 121X2P1 1.54 1.09 0.69 0.8 G588E 121X2P1 0.8 1.07 1.26 0.02 D684Y 121X2P1 1.24 1.03 1.64 0.85 S293Q 121X2P1 1.8 1 1.14 0.27 N298K/K374H 121X2P1 1.06 0.96 1.3 0.4 I638A 121X2P1 1.24 0.93 0.97 0.92 Q221M/N224R/K374H 121X2P1 1.22 0.92 1.26 0.35 G062E/D684M 121X2P1 1.34 0.89 1.6 0.19 S294T 121X2P1 1.17 0.88 1.3 0.33 D684M 121X2P1 1.55 0.82 1.65 0.73 S294T/G561D 121X2P1 1.18 0.81 1.01 0.16 K420N/D14D-Y 121X2P1 1.26 0.72 1.07 0.07 K374H 121X2P1 1.48 0.52 1.3 0.62 A547Y/T647M/K682V wild type 1 3.28 1.33 1.63 A547Y/T647M wild type 1.17 2 1.26 1.01 K522R/A547F/K682Y wild type 1.32 0.9 1.06 1.51 A547F/K682Y wild type 1.08 1.62 1.1 1.21 N224K/L549R wild type 0.47 2.39 1.21 0 N224K/F483N/L549R wild type 0.53 2.76 1.06 0 N535I/T560R/R632L wild type 0.52 3.03 1.08 0.05 Q221G/L549R/K655F wild type 1.27 3.11 1.16 0.01 Q221G/L549R wild type 0.51 1.51 1.08 0 I295W/K530T wild type 0.58 1.38 0.96 1.98 E475H/A547W/K655Y wild type 0.5 4.48 1.27 1.09 Q546W/K655F/S692F wild type 1.15 0.22 1.09 0.36 Y225T/S366A/T647F wild type 3.47 2.04 3.24 0.04 Y225T/S366A/T647F/K573M/L576M wild type 2.97 1.49 2.53 0 Y225T/S366A/T647F/K573M wild type 3.2 0.87 2.64 0.15 S366A/T647F wild type 0.85 0.19 1.19 0 I295W wild type 0.61 1.66 0.92 1.66 I295W/S369F wild type 0.78 3.3 0.92 1.05

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

CITATION LIST Patent Literature

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Non-Patent Literature

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What is claimed is:
 1. An isolated polypeptide having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity SEQ ID NO: 1, wherein the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 comprises a V299Y/R/E/A/F/K substitution relative to SEQ ID NO:
 2. 2. The isolated polypeptide of claim 1, wherein said isolated polypeptide further has a substitution corresponding to Y242A.
 3. The isolated polypeptide of claim 1, wherein said isolated polypeptide further has a substitution corresponding to E585K.
 4. The isolated polypeptide of claim 1, wherein said isolated polypeptide further has substitutions corresponding to Y242A and E585K.
 5. The isolated polypeptide of claim 1, comprising a set of substitutions selected from the group consisting of: T529M+S366A+A547F+N545L+Y225L+D657R+S692F+V299Y/R/E/A, T529M+8366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+V299A/R, T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+D693P+V299F, and T529M+S366A+A547F+N545L+Y225L+D657R+K561G+K541R+T544A+S692Y+I652Q+D684M+V299R.
 6. A method for sequencing a nucleic acid sample with the aid of an alpha-hemolysin nanopore in a membrane adjacent to a sensing electrode, the method comprising: a. providing tagged nucleotides into a reaction chamber comprising said nanopore, wherein an individual tagged nucleotide of said tagged nucleotides contains a tag coupled to a nucleotide, which tag is detectable with the aid of said nanopore; b. carrying out a polymerization reaction with the aid of a single isolated polypeptide according to claim 1, said isolated polypeptide being coupled to the nanopore, thereby incorporating an individual tagged nucleotide of said tagged nucleotides into a growing strand complementary to a single stranded nucleic acid molecule from said nucleic acid sample; and c. detecting, with the aid of said nanopore, a tag associated with said individual tagged nucleotide during incorporation of said individual tagged nucleotide, wherein said tag is detected with the aid of said nanopore while said nucleotide is associated with said variant polymerase.
 7. A composition comprising the isolated polypeptide of claim 1 covalently linked to an alpha-hemolysin monomer.
 8. The composition of claim 7, wherein the alpha-hemolysin monomer to which the isolated polypeptide is covalently linked is a constituent of a heptamer nanopore.
 9. The composition of claim 8, wherein the heptamer nanopore comprises 6 alpha-hemolysin monomers that are not covalently attached to the isolated polypeptide and one alpha-hemolysin monomer that is attached to the isolated polypeptide.
 10. The isolated polypeptide of claim 1, wherein the amino acid sequence of the isolated polypeptide has at least 95% sequence identity to SEQ ID NO: 2 when aligned using CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.
 11. The isolated polypeptide of claim 1, wherein the amino acid sequence of the isolated polypeptide has at least 98% sequence identity to SEQ ID NO: 2 when aligned using a CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.
 12. The isolated polypeptide of claim 1, wherein said isolated polypeptide is capable of catalyzing template-directed synthesis of a DNA polynucleotide. 